Staining of the cells in PEalphaDC80 in PBS + 2 % FCS

Day 3: change medium and start selection for stably transduced cells

Day 2: remove the medium from the cells. Mix the medium containing virus gently by pipetting and add to the cells (+ 6 migrograms/ml of polybrene). Incubate the cells at 37°C overnight.

Day 1: seed cells

Day 4: harvesting (virus-containing)

Day 3: change of medium

Day 2: transfection

Day 1: 4 x 10^6 293 FT in 10 ml DMEM (+10 % FCS (Tet-frei), +P/S) in a 10cm plate

Fc-block for 30 min on ice

Incubate cells in Fc-block (1:50 in PBS + 2 % FCS) 30 min on ice

Trypsinize cells and fix in 4 % PFA (or formalin)

Take supernatant and freeze at -80°C for analysis of DC-stimulation

Coculture of DCs and cells to be taken up (1(-2)x10^5 + 1(-2)x10^5 cells in 12-well) for 4 h to 24 h at 37°C

OR

After peptide pulsation cells are diluted in medium containing 20 ng/mL TNF-?, plus IL-4 and GM-CSF as stated above.

2 x 200 x 10e6 monocytes are transferred into culture bags by sterile welding and cultured for 5 days at a density of 2.5-5 x 10e6 cells/mL in serum free medium (CellGro, Cell Genix) in the presence of 100 ng/mL GM-CSF (Leukine, Berlex) and 20 ng/mL IL-4 (Cell Genix). After 2 days one volume fresh medium containing 2x concentrated cytokines is added.

The majority of monocytes (1 x 10e9) and all lymphocytes (6,4 x 10e9) are frozen in 90% A-plasma and 10% GMP-grade DMSO.

A HLA-A2 positive healthy donor undergoes leukapheresis.